Molecular studies on a novel lipoprotein from Mycobacterium bovis BCG

Vosloo, Wilna

Molecular studies on a novel lipoprotein from Mycobacterium bovis BCG

Thesis / Dissertation

1998

Publisher

University of Cape Town

Department

Division of Medical Microbiology

Faculty

Faculty of Health Sciences

Abstract

The Mycobacterium bovis BCG mpb83 gene was characterised in this study. It was shown that the gene was present in the Mycobacterium tuberculosis complex organisms as well as in Mycobacterium kansasii. The expression of the gene was variable as only a few isolates expressed the gene product at detectable levels. No definite sequence changes were observed outside the gene coding region that could lead to modulation or a change in regulation. The mpb70 and mpb83 genes were found to be adjacent to each other on the BCG Tokyo and M. tuberculosis H37Rv genomes. The two genes were not transcribed from a polycistronic mRNA. The transcription start site of mpb83 was determined, providing further evidence that mpb83 is transcribed as a single unit. A possible activator site, active in Mycobacterium smegmatis, was identified in the promoter region upstream of mpb83. Protein for M. smegmatis cell extract bound to this DNA region. However, DNA-protein interactions were different with lysates from fast-growing and slow-growing mycobacterial species. A construct that caused overexpression of MPB83 in M. smegmatis cells was also characterised, and it was shown that a cloning artefact led to the creation of a more efficient -1 O promoter region. Regulation of expression was also investigated in vitro and it was found that the expression of MPB83 in M. tuberculosis H37Rv was upregulated by stress conditions, viz. heat shock and a reducing medium. The signal peptide of MPB83 contains a lipoprotein cleavage site, and it was demonstrated that MPB83 was recognised and processed as a lipoprotein in homologous as well as in heterologous host. A mutation in the cleavage site abolished lipoprotein recognition.

Keywords

mycobacterium bovis

lipoprotein

Reference:

Collections

PhD / Doctoral

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